Blair Kris M, Bohinc Dillon J, Bane Kara L, Warnock Mark, Abuaita Basel, Gura Colby, Grinsztejn Eduarda, Marshall Steven H, Wilson Brigid M, Bonomo Robert A, Tambralli Ajay, Knight Jason S, O'Riordan Mary X, Lawrence Daniel A, Stavrou Evi X, Sandkvist Maria
Department of Microbiology and Immunology (K.M.B., B.A., C.G., M.X.O., M.S.), University of Michigan Medical School, Ann Arbor.
Presently at Fred Hutch/University of Washington/Seattle Children's Cancer Consortium, Louisiana State University School of Veterinary Medicine, Baton Rouge (K.M.B.).
Circ Res. 2025 Jun 20;137(1):e1-e15. doi: 10.1161/CIRCRESAHA.124.324764. Epub 2025 May 13.
FXII (coagulation factor XII) is best known for its roles in the contact and kallikrein-kinin pathways. FXII is converted to FXIIa (activated factor XII) by PKa (plasma kallikrein) or its unique ability to autoactivate on bacterial or other biologic surfaces. In vivo, FXIIa initiates the intrinsic coagulation pathway and promotes inflammation by reciprocal activation of prekallikrein, which cleaves HK (high-molecular-weight kininogen) to liberate bradykinin. CpaA (coagulation targeting metallo-endopeptidase of ) is a secreted metalloprotease identified in a human clinical isolate of that cleaves FXII at O-linked glycosylated sites, inhibiting contact activation. While CpaA facilitates a modest in vivo fitness advantage in mice, the role of CpaA in human infection remains unclear. As such, the objectives of this study were to characterize the structural details of the interaction between CpaA, FXII, and the KKS (kallikrein-kinin system) and to determine the downstream consequences on thromboinflammatory responses.
The effect of purified CpaA on the coagulant activity of FXII and the generation of bradykinin was characterized. Neutrophil signaling, flow cytometry, and functional assays were performed to define how CpaA-mediated cleavage of FXII affects innate immune functions. Bacterial killing by human neutrophils was performed with wild-type and mutant strains lacking CpaA.
We found that CpaA cleaves both FXII zymogen and FXIIa but not beta Factor XII. However, cleavage of FXIIa by CpaA does not significantly inhibit its clotting activity, demonstrating that CpaA does not inactivate FXIIa, but rather prevents activation of zymogen FXII. CpaA also cleaves HK, resulting in reduced kallikrein activation and bradykinin generation. We previously identified that zymogen FXII interacts with the urokinase receptor on neutrophils and upregulates neutrophil activation. Here, we demonstrate that CpaA cleaves neutrophil FXII, resulting in reduced Akt2 phosphorylation, chemotaxis, oxidative burst, and neutrophil extracellular trap formation. Importantly, CpaA decreases the human neutrophil killing efficiency of in culture.
These data identify a role for FXII in responding to bacterial infection and suggest that by inhibiting the contact and kallikrein-kinin pathways and impairing neutrophil activation, CpaA may blunt the innate immune response and help prevent the elimination of from the human host.
凝血因子 XII（FXII）因其在接触途径和激肽释放酶 - 激肽途径中的作用而最为人所知。FXII 通过血浆激肽释放酶（PKa）转化为活化的凝血因子 XII（FXIIa），或者凭借其在细菌或其他生物表面上自动激活的独特能力进行转化。在体内，FXIIa 启动内源性凝血途径，并通过前激肽释放酶的相互激活促进炎症反应，前激肽释放酶可裂解高分子量激肽原（HK）以释放缓激肽。CpaA（凝血靶向金属内肽酶）是在一种人类临床分离株中鉴定出的分泌型金属蛋白酶，它在 O - 连接糖基化位点裂解 FXII，从而抑制接触激活。虽然 CpaA 在小鼠体内具有适度的适应性优势，但其在人类感染中的作用仍不清楚。因此，本研究的目的是表征 CpaA、FXII 和激肽释放酶 - 激肽系统（KKS）之间相互作用的结构细节，并确定对血栓炎症反应的下游影响。
表征纯化的 CpaA 对 FXII 凝血活性和缓激肽生成的影响。进行中性粒细胞信号传导、流式细胞术和功能测定，以确定 CpaA 介导的 FXII 裂解如何影响先天免疫功能。使用野生型和缺乏 CpaA 的突变菌株进行人类中性粒细胞的细菌杀伤实验。
我们发现 CpaA 可裂解 FXII 酶原和 FXIIa，但不裂解β凝血因子 XII。然而，CpaA 对 FXIIa 的裂解并未显著抑制其凝血活性，这表明 CpaA 不会使 FXIIa 失活，而是阻止 FXII 酶原的激活。CpaA 还可裂解 HK，导致激肽释放酶激活减少和缓激肽生成减少。我们之前发现 FXII 酶原与中性粒细胞上的尿激酶受体相互作用并上调中性粒细胞激活。在此，我们证明 CpaA 可裂解中性粒细胞中的 FXII，导致 Akt2 磷酸化、趋化性、氧化爆发和中性粒细胞胞外陷阱形成减少。重要的是，CpaA 降低了培养物中人类中性粒细胞对细菌的杀伤效率。
这些数据确定了 FXII 在应对细菌感染中的作用，并表明通过抑制接触途径和激肽释放酶 - 激肽途径以及损害中性粒细胞激活，CpaA 可能会减弱先天免疫反应，并有助于防止人类宿主清除细菌。
